For polyamine analysis, 150 mg of leaf or root material was harvested from plants grown in the greenhouse at different stages of development: before flowering (leaves 6 and 12, numbered from base), at topping (leaves 19 and 23 and roots), and at harvest (leaves 23 and 24 and roots). Samples were collected, after 4 hr of illumination and were flash frozen in liquid nitrogen. For time‐course monitoring of total polyamine content, six plants were used for collection of root samples from PMT‐RNAi plants and eight WT, PR50‐RNAi, and PMT‐RNAi plants for collection of leaf and/or root samples. For analysis of free and conjugated polyamines, four biological replicates were used. Extraction of free and conjugated polyamines was performed as previously described (Nölke et al., 2018 ). The dansilation of free and conjugated polyamines was carried out with dansyl chloride as described by Flores and Galston (1982 ). The dansylated polyamines were measured by LC‐MS/MS. All experiments were carried out on a 3200 QTRAP™ mass spectrometer (Sciex) coupled to an HPLC Agilent 1200 system as described before (Nölke et al., 2018 ).
3200 qtrap mass spectrometer
Manufactured by AB Sciex
Sourced in United States, Germany, Canada
The 3200 QTRAP mass spectrometer is a highly sensitive and versatile analytical instrument designed for a wide range of applications. It combines the capabilities of a triple quadrupole mass spectrometer with the functionality of a linear ion trap, providing users with enhanced sensitivity, specificity, and flexibility in their research and analysis workflows. The core function of the 3200 QTRAP is to accurately measure and identify a diverse range of compounds, enabling researchers and analysts to gather critical data for their applications.
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Lab products found in correlation
Agilent 1200 series hplc system, by Agilent Technologies (8 mentions)
Zorbax sb c18 column, by Agilent Technologies (5 mentions)
Analyst 1, by AB Sciex (5 mentions)
Agilent 1200 series hplc, by Agilent Technologies (2 mentions)
Luna rp c5 column, by Phenomenex (2 mentions)
Bca assay kit, by Thermo Fisher Scientific (2 mentions)
Zorbax sb c18 analytical column, by Agilent Technologies (2 mentions)
Multiquant 2, by AB Sciex (2 mentions)
Agilent 1200 hplc system, by Agilent Technologies (2 mentions)
1200 series hplc system, by Agilent Technologies (2 mentions)
Nexera x2 lc 30ad, by Shimadzu (1 mentions)
Qtrap 6500, by AB Sciex (1 mentions)
Pge2 d4, by Cayman Chemical (1 mentions)
Aea d4, by Cayman Chemical (1 mentions)
2 ag d5, by Cayman Chemical (1 mentions)
Oea d4, by Cayman Chemical (1 mentions)
Aa d8, by Cayman Chemical (1 mentions)
Nexera system, by AB Sciex (1 mentions)
Pgf2α, by Cayman Chemical (1 mentions)
Pgd2 d4, by Cayman Chemical (1 mentions)
47 protocols using 3200 qtrap mass spectrometer
1
Polyamine Analysis in Plant Tissues
2
Quantitative Metabolite Extraction and Analysis
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The cell samples were thawed on ice, vortexed, and freeze-thawed three times and vortexed to completely break the cells. Then we took a 100 µl sample and added 10 L internal standard, vortexed for 10 s, added 500 L ethyl acetate, and vortexed for 30 s. After concentration centrifugation at 14,000 × g for 5 min, 400 µl of supernatant was collected for LC-MS/MS analysis. A NexeraX2 LC-30AD (Shimadzu) chromatograph system coupled with a 3200 QTRAP mass spectrometer (Sciex) was used for quantitative analysis. An Atlantis T3 C18 column (50 mm × 2.1 mm I.D.3.5 µm) was used and the column oven temperature was 40 °C. The mobile phase was composed of solvent A (H2O) and B (acetonitrile), both containing 0.1 % formic acid. The gradient was as follows: 0–1 min, 5% B; 1–1.2 min, 5% B; 1.2–3.0 min, 95% B; 3.0–3.2 min, 95% B; and 3.2–4.0 min, 5% B for column re-equilibration.
3
Quantifying Phenolic Compounds in Foods
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Phenolic acids and flavonoid aglycones were determined with reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry (LC-ESI-MS/MS).
The Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary gradient solvent pump, a degasser, an autosampler, and a column oven connected to a 3200 QTRAP Mass spectrometer ((Sciex, Redwood City, CA, USA) was used.
The contents of phenolic acids and free flavonoid aglycones were determined with a simple and rapid method using liquid chromatography–electrospray ionization–tandem mass spectrometry. The compounds were separated at 25 °C on a Zorbax SB-C18 column (2.1 × 50 mm, 1.8-μm particle size; Agilent Technologies, Santa Clara, CA, USA).
The contents of phenolic acids were determined with the LC-ESI-MS/MS method described previously by Nowacka et al. (2014) [44 (link)] with some modifications.
The determination of flavonoid aglycones was carried out using the LC-ESI-MS/MS method described by Pietrzak et al. (2017) [45 (link)] with some modifications.
The Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary gradient solvent pump, a degasser, an autosampler, and a column oven connected to a 3200 QTRAP Mass spectrometer ((Sciex, Redwood City, CA, USA) was used.
The contents of phenolic acids and free flavonoid aglycones were determined with a simple and rapid method using liquid chromatography–electrospray ionization–tandem mass spectrometry. The compounds were separated at 25 °C on a Zorbax SB-C18 column (2.1 × 50 mm, 1.8-μm particle size; Agilent Technologies, Santa Clara, CA, USA).
The contents of phenolic acids were determined with the LC-ESI-MS/MS method described previously by Nowacka et al. (2014) [44 (link)] with some modifications.
The determination of flavonoid aglycones was carried out using the LC-ESI-MS/MS method described by Pietrzak et al. (2017) [45 (link)] with some modifications.
4
Quantitative Lipid Analysis by LC-MS/MS
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HPLC-grade methanol, acetonitrile, isopropanol and formic acid used for sample purification and LC-MS/MS analysis were JT Baker-brand (ThermoFisher Scientific, Waltham, MA). Authentic lipid standards of 6-keto-PGF1α, PGE2, PGD2, PGF2α, PGJ2, 15-hydroxyeicosanoic acid (15-HETE), 2-arachidonoylglycerol (2-AG), anandamide (AEA), arachidonic acid (AA), oloeylglycerol (OG), and oleoylethanolamide (OEA) were purchased from Cayman Chemicals (Ann Arbor, MI). 16:0 lysophosphatidylcholine (LPC) and 17:0 LPC were purchased from Avanti Polar Lipids (Alabster, AL). The following deuterated internal standards were purchased from Cayman: 6-keto-PGF1α-d4, PGF2α-d4, PGE2-d4, PGD2-d4, PGJ2-d4, 15-HETE-d8, 2-AG-d5, AEA-d4, AA-d8, and OEA-d4. OG-d5 was purchased from US Biological (Salem, MA).
All LC-MS/MS analysis was performed on a Shimadzu Nexera system in-line with a SCIEX 6500 QTrap; except the LPC analysis, which was performed on a Shimadzu LC system in-line with a SCIEX 3200 QTrap mass spectrometer. The 6500 QTrap was equipped with a TurboV Ionspray source and operated in positive and negative ion modes. The 3200 QTrap was equipped with an ionspray source and operated in positive ion mode. For both LC-MS systems, SCIEX Analyst software (ver 1.6.2) was used to control the instruments and acquire and process the data.
All LC-MS/MS analysis was performed on a Shimadzu Nexera system in-line with a SCIEX 6500 QTrap; except the LPC analysis, which was performed on a Shimadzu LC system in-line with a SCIEX 3200 QTrap mass spectrometer. The 6500 QTrap was equipped with a TurboV Ionspray source and operated in positive and negative ion modes. The 3200 QTrap was equipped with an ionspray source and operated in positive ion mode. For both LC-MS systems, SCIEX Analyst software (ver 1.6.2) was used to control the instruments and acquire and process the data.
5
Benzodiazepine Analysis by IL-DLLME-LC-MS/MS
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A Shimadzu Prominence Ultra-Fast Liquid Chromatograph XR System (Shimadzu Benelux, Jette, Belgium) equipped with a Kinetex ® Biphenyl LC Column (100 mm × 2.1 mm, 2.6 m particle size) (Phenomenex, Utrecht, The Netherlands) was used. The LC system was coupled to a 3200 QTRAP mass spectrometer (Sciex, Halle, Belgium), operated in scheduled multiple reaction monitoring (sMRM) scan mode.
For a detailed description of the used IL-DLLME-LC-MS/MS method for benzodiazepine analysis, the reader is referred to the ME values were calculated for both sample sets (whole blood and Milli-Q water extracts) and compared by means of a multiple t-test analysis (␣ = 0.05). Significantly lower ME values for whole blood extracts would indicate that interfering blood matrix components were not sufficiently removed using IL-DLLME. Additionally, coefficients of variation (CVs) (n = 3) were calculated and the acceptance limit was ≤ 15%.
For a detailed description of the used IL-DLLME-LC-MS/MS method for benzodiazepine analysis, the reader is referred to the ME values were calculated for both sample sets (whole blood and Milli-Q water extracts) and compared by means of a multiple t-test analysis (␣ = 0.05). Significantly lower ME values for whole blood extracts would indicate that interfering blood matrix components were not sufficiently removed using IL-DLLME. Additionally, coefficients of variation (CVs) (n = 3) were calculated and the acceptance limit was ≤ 15%.
6
LC-MS/MS Quantification of Phosphatidylethanol
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The LC-MS/MS system consisted of a CTC PAL autosampler (CTC Analytics, Zwingen, Switzerland), an Agilent 1200 series HPLC (Agilent, Waldbronn, Germany) and a 3200 QTrap mass spectrometer (Sciex, Toronto, Canada) controlled by Analyst™ software (version 1.5.1).
Analytical separation was performed by a Luna RP-C5 column, 50 mm×2 mm, 5 μm (Phenomenex, Brechbühler, Schlieren, Switzerland) heated to 50 °C with a flow rate of 0.3 mL/min. Mobile phase A consisted of ammonium acetate (2 mM)/acetonitrile (30:70, v/v) solution, and mobile phase B was 2-propanol. The following 10-min gradient was used: 0 to 1.5 min, 10 % B; 1.5 to 2.5 min, 10 to 40 % B linear; 2.5 to 3.5 min, 40 to 100 % B linear; 3.5 to 4.5 min, 100 % B; 4.5 to 6 min, 100 to 10 % B linear; and 6 to 10 min, 10 % B. Postcolumn infusion of 2-propanol (0.3 mL/min) was used to increase the signal intensity.
The mass spectrometer was operated in negative ESI MRM mode, with an ion-spray voltage of -4250 V and a source temperature of 650 °C with the following transitions for PEth 16:0/18:1: m/z 701.5/255.1 (quantifier), m/z 701.5/ 281.1 and m/z 701.5/437.2 (qualifiers), and m/z 706.5/281.1 (D 5 -PEth 16:0/18:1). For PEth 16:0/18:2, the transitions were the following: m/z 699.5/255.2 (quantifier), m/z 699.5/279.2 and m/z 699.5/437.2 (qualifiers), and m/z 704.5/279.4 (D 5 -PEth 16:0/18:2).
Analytical separation was performed by a Luna RP-C5 column, 50 mm×2 mm, 5 μm (Phenomenex, Brechbühler, Schlieren, Switzerland) heated to 50 °C with a flow rate of 0.3 mL/min. Mobile phase A consisted of ammonium acetate (2 mM)/acetonitrile (30:70, v/v) solution, and mobile phase B was 2-propanol. The following 10-min gradient was used: 0 to 1.5 min, 10 % B; 1.5 to 2.5 min, 10 to 40 % B linear; 2.5 to 3.5 min, 40 to 100 % B linear; 3.5 to 4.5 min, 100 % B; 4.5 to 6 min, 100 to 10 % B linear; and 6 to 10 min, 10 % B. Postcolumn infusion of 2-propanol (0.3 mL/min) was used to increase the signal intensity.
The mass spectrometer was operated in negative ESI MRM mode, with an ion-spray voltage of -4250 V and a source temperature of 650 °C with the following transitions for PEth 16:0/18:1: m/z 701.5/255.1 (quantifier), m/z 701.5/ 281.1 and m/z 701.5/437.2 (qualifiers), and m/z 706.5/281.1 (D 5 -PEth 16:0/18:1). For PEth 16:0/18:2, the transitions were the following: m/z 699.5/255.2 (quantifier), m/z 699.5/279.2 and m/z 699.5/437.2 (qualifiers), and m/z 704.5/279.4 (D 5 -PEth 16:0/18:2).
7
Choline Analysis in Pup Stomach
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Stomach contents of pups were analyzed to reflect the choline concentration in the pup’s diet during lactation. Splenocytes and frozen stomach content were extracted using a modified Bligh and Dyer method that has been previously described in [21 (link),22 (link)]. Extracts were quantified for all significant choline-containing metabolites and total choline content by HILIC liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Agilent 1200 series HPLC system (Agilent Technologies, Palo Alto, CA, USA) coupled to a 3200 QTRAP mass spectrometer (AB SCIEX, Concord, ON, Canada). The choline content in splenocytes was adjusted for protein content, which was measured using a commercial bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Edmonton, AB, Canada) according to the manufacturer’s instructions.
8
Quantification of Flavonoids and Phenolic Acids in Hemerocallis Extracts
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Agilent 1200 Series HPLC system (Agilent Technologies, Palo Alto, CA, USA) coupled to 3200 QTRAP mass spectrometer (AB Sciex, Redwood City, CA, USA) was used for qualitative and quantitative analysis of flavonoids and phenolic acids in Hemerocallis extracts. The separation of analyzed compounds, injected in a 3-µL amount, was performed on a Zorbax SB-C18 analytical column (2.1 × 100 mm, 1.8 µm, Agilent Technologies, Palo Alto, CA, USA) at 25 °C. Elution was carried out using solvent A (0.1% HCOOH in water) and solvent B (0.1% HCOOH in acetonitrile). The following gradient elution program was used: 0–2 min—20% B, 3–4 min—25% B, 5–6 min—35% B, 5–6 min—35% B, 8–12 min—65% B, 14–16 min—80% B, 20–28 min—20% B. The flow rate was 300 µL/min. The mass spectra of analyzed compounds were acquired in the negative ESI mode, and the optimum values of the source parameters were as follows: capillary temperature 450 °C, nebulizer gas 50 psi, curtain gas 30 psi, source voltage −4500 V for phenolic acids and flavonoid glycosides, and capillary temperature 550 °C, nebulizer gas 30 psi, curtain gas 20 psi, and source voltage −4500 V for flavonoid aglycones analysis. Details of LC-ESI-MS/MS analysis are presented in Table 1 and were described in our previous research [41 (link)]. The Analyst 1.5 software (AB Sciex, Redwood City, CA, USA) was used for analysis and data acquisition.
9
Rhamnolipid Characterization by ESI-MS
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Electrospray ionization mass spectra were recorded on a 3200 QTRAP mass spectrometer, AB SCiex. A 3000 ppm stock solution of rhamnolipid was prepared with ultra-pure water as the solvent. The concentration of the sample was 20 ppm and a methanol/water (1:1) + 0.1% of ammonia hydroxide solution was used as the solvent. The sample was introduced into the source at a rate of 10 μl/min with a syringe pump. The spectra were acquired in the negative mode in a mass range 100–1000 m/z using the following conditions: −4500 V, Curtain Gas:10 psi, Gas 1:15 psi, DP (Declustering Potential): −20.0 V, EP (Entrance Potential): −7.0 V e CEP (Cell entrance potential): −12.0 V. The fragment ion experiment (MS2) was performed using a collision energy of 35.0 V +/− 15.0 V.
10
LC-MS/MS Quantification of Compounds
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The system
was composed of a Shimadzu Prominence LC20 system (Shimadzu Corp.,
Kyoto, Japan) coupled to a 3200 Qtrap mass spectrometer (AB Sciex,
Ontario, Canada). The column used was a Supelco Discovery HS C18,
2.1 × 150 mm, 3 μm, and the mobile phases were A: 10% acetonitrile
in water with 0.1% formic acid and B: 100% acetonitrile with 0.1%
formic acid. The gradient elution program was at 0.15 mL/min and started
at 20% B, followed by a ramp to 100% B in 18 min and kept for 7.5
min, then reduced to 20% B in 0.5 min and kept at 20% B for 4 min.
The MS was operated in ESI+ using multiple reaction monitoring
(MRM). The collision energy was set to 50 V. Curtain gas was set to
40 and the ionization spray at +5000 V. The source temperature was
set to 450 and gases 1 and 2 were set to 20 and 15, respectively.
Declustering potential was set to 95.
was composed of a Shimadzu Prominence LC20 system (Shimadzu Corp.,
Kyoto, Japan) coupled to a 3200 Qtrap mass spectrometer (AB Sciex,
Ontario, Canada). The column used was a Supelco Discovery HS C18,
2.1 × 150 mm, 3 μm, and the mobile phases were A: 10% acetonitrile
in water with 0.1% formic acid and B: 100% acetonitrile with 0.1%
formic acid. The gradient elution program was at 0.15 mL/min and started
at 20% B, followed by a ramp to 100% B in 18 min and kept for 7.5
min, then reduced to 20% B in 0.5 min and kept at 20% B for 4 min.
The MS was operated in ESI+ using multiple reaction monitoring
(MRM). The collision energy was set to 50 V. Curtain gas was set to
40 and the ionization spray at +5000 V. The source temperature was
set to 450 and gases 1 and 2 were set to 20 and 15, respectively.
Declustering potential was set to 95.
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